Photosynthesis Research35: 191-200, 1993. ~) 1993 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Photosystem II reaction centres stay intact during low temperature photoinhibition Christina Ottander, Torill Hundal 1, Bertil Andersson 1, Norman P.A. Huner 2 & Gunnar Oquist

Department of Plant Physiology, University of Ume& S-901 87 Umegt, Sweden; ~Department of Biochemistry, Arrhenius Laboratories, University of Stockholm, S-106 91 Stockholm, Sweden; 2Department of Plant Sciences, University of Western Ontario, London, Ontario, N6A 5B7, Canada Received 10 April 1992; accepted in revised form 28 September 1992

Key words:

chlorophyll fluorescence, Dl-protein, fluorescence quenching, low temperature, photoinhibition, photosynthesis, Photosystem II Abstract

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibition at low temperature by 1) creation of excessive excitation of Photosystem II or, 2) inhibition of the repair process of Photosystem II. The light and temperature dependence of the reduction state of QA was measured by modulated fluorescence. Photon flux densities giving 60% of QA in a reduced state at steady-state photosynthesis (300 ~mol m -2 s -1 at 5°C and 1200 ~mol m -2 s -1 at 20°C) resulted in a depression of the photochemical efficiency of Photosystem II (Fv/Fm) at both 5 and 20 °C. Inhibition of Fv/F m occurred with initially similar kinetics at the two temperatures. After 6 h, Fv/F m was inhibited by 30% and had reached steady-state at 20 °C. However, at 5 °C, Fv/F m continued to decrease and after 10 h, Fv/F m was depressed to 55% of control. The light response of the reduction state of QA did not change during photoinhibition at 20 °C, whereas after photoinhibition at 5 °C, the proportion of closed reaction centres at a given photon flux density was 10-20% lower than before photoinhibition. Changes in the Dl-content were measured by immunoblotting and by the atrazine binding capacity during photoinhibition at high and low temperatures, with and without the addition of chloramphenicol to block chloroplast encoded protein synthesis. At 20 °C, there was a close correlation between the amount of Dl-protein and the photochemical efficiency of photosystem II, both in the presence or in the absence of an active repair cycle. At 5 °C, an accumulation of inactive reaction centres occurred, since the photochemical efficiency of Photosystem II was much more depressed than the loss of Dl-protein. Furthermore, at 5 °C the repair cycle was largely inhibited as concluded from the finding that blockage of chloroplast encoded protein synthesis did not enhance the susceptibility to photoinhibition at 5 °C. It is concluded that, the kinetics of the initial decrease of F J F m was determined by the reduction state of the primary electron acceptor QA, at both temperatures. However, the further suppression of Fv/F m at 5 °C after several hours of photoinhibition implies that the inhibited repair cycle started to have an effect in determining the photochemical efficiency of Photosystem II.

Abbreviations: CAP-D-threochloramphenicol; F 0 and F~-fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively; F m and F m-fluorescence when all Photosystem II reaction centres are closed in dark- and light-acclimated leaves, respectively;

192 F s-fluorescence at steady state; QA-the primary, stable quinone acceptor of Photosystem II; qN -- non-photochemical quenching of fluorescence; qp - photochemical quenching of fluorescence

Introduction

Photoinhibition of photosynthesis by high light is first seen as an inhibition of the photon yield of CO 2- uptake or O2-evolution (Powles 1984). Photosystem II is considered to be the primary site of photoinhibition, which is generally thought to occur in the reaction centre itself (Krause 1988). Furthermore, photoinhibition triggers degradation of the Dl-protein of the Photosystem II reaction centre (Kyle et al. 1984, Andersson et al. 1992). Alternatively, a disturbed energy transfer from the antenna to the reaction centre may also explain the inhibited yield of photosynthesis during photoinhibition (Demmig and Bj6rkman 1987). Photoinhibition of photosynthesis may occur under natural conditions in the absence of any stress other than high light (Ogren 1988). However, suboptimal environmental conditions may promote photoinhibition even when the photon flux densities are moderate (Osmond 1981). Low temperature is an important environmental stress which makes photosynthesis more sensitive to photoinhibition so that even low photon flux densities may cause photoinhibition (Oquist et al. 1987, Oquist and Huner 1991). There are at least four hypotheses to explain why plants become much more sensitive to photoinhibition at low temperatures. 1. Low temperatures would reduce the capacity of photosynthesis and thereby increase the probability for excessive excitation of Photosystem II. 2. The ability for repair of Photosystem II is reduced at low temperatures. Degradation and synthesis of the Dl-protein in the reaction centre appears to be slowed down by low temperatures (Chow et al. 1989, Gong and Nilsen 1989, Aro et al. 1990). Also, the rates of migration, protein assembly and ligation of co-factors are slowed down at low temperatures (Kyle 1987). 3. The capacity of oxygen scavengers, which have the potential of providing protection to photoinhibition, decreases at low temperature

(Richter et al. 1990, Sch6ner et al. 1989). 4. The ability to form zeaxanthin, which is thought to be able to quench excitation energy in the antenna of Photosystem II (Demmig and Bj6rkman 1987, Demmig-Adams et al. 1990), may be inhibited at low temperature (Bilger and Bj6rkman 1991). These hypotheses are not necessarily mutually exclusive. In earlier work (Greer et al. 1991) it was shown that the steady-state extent of photoinhibition was strongly dependent on temperature. It was proposed that the steady-state photochemical efficiency (Fv/Fm) established during photoinhibition was the result of different mechanisms at different temperatures. At 20 °C, net photoinhibition was determined by the difference between inactivation and repair of Photosystem II; i.e., the degradation and synthesis of the reaction centre protein, D1. At 5 °C, the repair process was largely inhibited, with increased photoinhibition as a consequence. It was suggested that some protection of remaining photochemicaUy active centres could be conferred by connected and stable photoinhibited reaction centres. The aim of this work was to determine whether this increased susceptibility to photoinhibition at low temperature was an effect of a decreased capacity of the Photosystem II repair cycle alone, or whether a temperature inhibition of photosynthesis causing increased excitation pressure of the reaction centre of Photosystem II also was involved. For this purpose we have exposed intact barley leaves to photoinhibitory conditions at 5 and 20 °C at equal proportion of reduced QA, i.e., equal excitation pressure. Modulated fluorescence has been used to monitor the excitation pressure on Photosystem II, as expressed by the reduction state of QA. To assay effects on the Photosystem II repair cycle, measurements of D1 content have been performed. The data presented supports the proposal that the steady-state level of photoinhibition reached at the different temperatures are determined by different mechanisms.

193 Material and methods

Plant material and photoinhibition treatments Barley (Hordeum vulgare L. var. Gunilla, Sval6f AB N83-4001) was grown in a climate chamber at a day/night temperature of 25/15 °C and a 12h photoperiod at a photon flux density of 200tzmol m - 2 s -1 provided by metal halide lamps (HQI-TS, 400 W, Osram Berlin, Germany). For photoinhibition, fully expanded primary leaves were bent and supported normal to the incident radiation and transferred to a second climate chamber containing a high-light treatment system of two extra metal halide lamps. Photon flux densities used during photoinhibition varied between 300-1200/~mol m - 2 s -~ Leaf temperature was maintained constant by the air circulation system within the chamber at either 5 or 20 °C and measured with a NiCr/NiA1 type K thermocouple. In some experiments, leaves were painted with 300/~g ml-1 CAP (Sigma Chemical Co., St Louis, MO, USA) in 1% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma) prior to transfer to photoinhibitory light.

Measurements of photosynthetic activity The photochemical efficiency of Photosystem II was measured as the Fv/F m ratio of dark-adapted leaves (30 rain) by room temperature chlorophyll fluorescence at 695 nm with a PSM fluorometer (Biomonitor S.C.I. AB Ume& Sweden). The reduction state of QA at steady-state photosynthesis, was measured with a pulse modulated fluorometer (PAM 101 and 103 with Schott lamp FL-103; H. Waltz, Effeltric, Germany), in a cuvette ventilated with water-saturated ambient air and with water-saturated gas mixture of 5% CO 2 in air and kept at 5 and 20 °C. Actinic light was provided from a Schott lamp (FL-101). Terminology and calculation of fluorescence parameters were applied according to van Kooten and Snel (1990). The maximal fluorescence in the presence of the actinic light, F', was determined using a flash of white light with a photon flux density of 16000/xmol m 2 s-~ and 1 s in length, which was enough light to fully close Photosystem II as tested by the Markgraf and Berry

method (1990). Far-red illumination of 8 W m -2 ( R G 715; Schott AG, Mainz, Germany) was used to get the fluorescence minimum, taken as F 0, corresponding to the state where the Photosystem II acceptor QA is re-oxidized. The reduction state of QA, or the fraction of closed Photosystem II centres, was estimated from Qred/ Qtot,~ = (Fs-F6)/(F'-F0) as described by Dietz et al. (1985). The reduction state of QA was used as an approximation of the excitation pressure on Photosystem II (Havaux et al. 1991). O2-evolution and reduction state of QA was measured simultaneously in a leaf disc electrode (LD 2; Hansatech Ltd., Kings Lynn, UK), ventilated with a water-saturated gas mixture of 5% C O 2 in air at 5 and 20 °C as described by Ogren (1988). The photosynthetic photon flux density was measured with a photon sensor (LI 185A, Lambda instruments co., Lincoln, Nebraska, USA).

Protein determinations Proteins were extracted by grinding barley leaves at 77 K, according to Nechushtai and Nelson (1985). The extraction buffer contained, 0.1M Tris-HCl, pH 6.8, 2 mM EDTA, 2% SDS, 2% /3-mercaptoethanol, 10% glycerol and protease inhibitors (5/zg/ml leupeptin, 0.01 mM pepstain and 2 mM phenylmethylsulphonylfluoride). SDSpolyacrylamide gel electrophoresis was carried out according to Laemmli (1970) using a 12 to 22.5% polyacrylamide gradient and 6 M urea in the separation gel. Changes in the relative D1protein content was determined by Western blotting (Towbin et al. 1979), using antibodies against the Dl-protein and the nuclear encoded 22 kDa protein. Protein A labelled with 125I was used to detect the antibodies. To quantify the results, autoradiograms were scanned with a laser densitometer. Determinations of 14C-atrazine binding to the Dl-protein were made according to Tischer and Strotmann (1977). Resuspended thylakoids diluted to 48/xg Chlm1-1 and 20/~1 of [ethyl-114C] atrazine (925 kBq /xmo1-1) were mixed to obtain a final concentration of 1/zM atrazine. The total reaction volume was 1.0 ml. The thylakoid mixtures were incubated for 10min under laboratory light conditions of 2/xmol m - 2 s i

194 and then centrifuged for 3 min at 16 000 g and 5°C. The supernatant (0.7ml) was added to 10 ml of Ecoscint (Diamed, Missisauga, Canada) and radioactivity determined in a Beckman scintillation counter (Model LS6000IC). The amount of labelled atrazine bound to the thylakoids was calculated from the difference between controls (without thylakoids) and corresponding thylakoid samples. Chlorophyll concentrations were determined according to Arnon (1949).

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Photon Flux Density (llmol m'2 s -1 ) Fig. 2. (A) Light response curves of the reduction state of the primary electron acceptor QA at steady-state photosynthesis at 20 (open) and 5 °C (filled) in intact harley leaves. Measurements were done in 5% CO 2 in water saturated air. (B) Light response curves of non-photochemical quenching (q~) of fluorescence at 20 (open) and 5 °C (filled). Bars show the standard deviation, n = 3-6.

The light response of the reduction state of Q A showed that Q A w a s more sensitive to irradiance at 5 than at 20 °C, with 50% closure of reaction centres at a PFD of 180 and 700 p~mol m - z s - 1 at 5 and 20°C, respectively (Fig. 2A). Consequently, the depression of photosynthesis observed at the lower temperature resulted in a significant increase in the proportion of reduced QA" At 5 °C, the non-photochemical quenching component (qN) reached light saturation of 0.90 already at 300/~mol photons m -z s -1 (Fig. 2B). At 20°C, a maximal qN-value of 0.86 was reached at a PFD of 1400 p~mol photons m - z s - 1 Thus, the higher proportion of open reaction centres as well as the lower values of qy at 20 °C, are probably explained by the higher rate of photosynthesis at 20 than at 5 °C. Since it has been shown that the susceptibility of photosynthesis to photoinhibition increases with the level of steady state reduction of Q A

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(Ogren 1991, (3quist et al. 1992), we examined the relative susceptibility to photoinhibition at 5 and 20°C by adjusting the light so that similar values of the reduction state of QA were maintained at the two temperatures. Photon flux densities causing a 60% steady-state reduction of QA in ambient air conditions, (i.e. 300 and 1200/~mol m -2 s -~ at 5 and 20°C, respectively, Fig. 3A, B) were chosen to study the time course of the decrease in Fv/F m (Fig. 4). During the first 6 h under conditions of equivalent initial reduction state of QA, the kinetics of the inhibition of the photochemical efficiency of Photosystem II (Fv/Fm) were similar and Fv/F m was inhibited 30% at both 5 and 20 °C (Fig. 4). However, after 6h, a steady-state level of photoinhibition was reached at 20°C, but Fv/F m continued to decrease at 5 °C. After 10 h, Fv/F m was inhibited 25 and 54% at 20 and 5 °C, respectively. To further evaluate the significance of the reduction state of QA in determining the suscep-

tibility to photoinhibition at the two temperatures, we compared the level of inhibition of Fv/F m after photoinhibitory treatment at different reduction state of QA, both with and without blocking repair of the Dl-protein. Figure 5 shows the extent of photoinhibition after 2.5 h treatment at different reduction state of Q A a t 5 and 20°C with and without addition of Dthreochloramphenicol (CAP, an inhibitor of chloroplast encoded protein synthesis). At both 5 and 20°C, with repair of PSII going on, the susceptibility of photosynthesis to photoinhibition was similarly controlled by the reduction

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196 state of QA (Fig. 5). Blocking repair of the Dl-protein resulted in a severe inhibition of Fv/F m at 20 °C, but at 5 °C, almost no increase in sensitivity occurred (Fig. 5, Greer et al. 1991). This indicates that the repair cycle is largely inactivated at 5 °C and thus has a very limited protective role at low temperatures. Furthermore, the steady-state reduction state of QA and the photochemical efficiency of Photosystem II after photoinhibition at 5 and 20 °C showed a curvilinear relationship (Fig. 6). At 5°C, measurements of Fv/F m were done after both 4 and 10 h of photoinhibition since more than one factor seemed to determine the decrease in Fv/F m (Fig. 4). The relation was similar after the different treatments but after 10h of photoinhibition at 5 °C a somewhat more pronounced decrease in Fv/F m was seen at a given reduction state of QA. Accordingly, photoinhibition for 10 h resulted in a somewhat larger fraction of the photochemically active reaction centres in an open configuration (Fig. 6). Since the reduction in Fv/F m followed the same kinetics at the two temperatures during the first six hours of photoinhibition (Fig. 4), but subsequently differed, it was of interest to see if the reduction state of QA changed during photoinhibition or if this difference was due to a temperature effect on the repair cycle. There was no change in the light response of the proportion of reduced QA after 6-10h of photoinhibition at 20°C (Fig. 3A). However, after

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6-8 h of photoinhibition at 5 °C, the proportion of reduced QA was 10-20% lower than before photoinhibition (Fig. 3B). Consequently, an accentuated temperature effect, increasing the proportion of closed Photosystem II reaction centres, cannot account for the further increase in susceptibility to photoinhibition at low temperature observed after 6 h of photoinhibition.

Effect of low temperature on the repair processes of Photosystem H Previous measurements have shown that recovery from photoinhibition was completed within 6 h at 25 °C in low light (

Photosystem II reaction centres stay intact during low temperature photoinhibition.

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibitio...
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